Cell-wall degrading enzyme variants

Abstract

The present invention relates to variants of a cell-wall degrading enzyme having a beta-helix structure, which variant has at least one substituent in a position determined by identifying all residues potentially belonging to a stack; characterizing the stack as interior or exterior; characterizing the stack as polar, hydrophobic or aromatic/heteroaromatic based on the dominating characteristics of the parent or wild-type enzyme stack residues and/or its orientation relative to the beta-helix (interior or exterior); optimizing all stack positions of a stack either to hydrophobic aliphatic amino acids, hydrophobic aromatic or polar amino acids by allowing mutations within one or all positions to amino acids belonging to one of these groups; measuring thermostability of the variants by DSC or an application-related assay such as a Pad-Steam application test; and selecting the stabilized variants. Variant of a wild-type parent pectate lyase (EC 4.2.2.2) having the conserved amino acid residues D111, D141 or E141, D145, K165, R194 and R199 when aligned with the pectate lyase comprising the amino acid sequence of SEQ ID NO: 2 are preferred.

What is claimed is:  
     
       1. A variant of a wild-type parent pectate lyase (EC 4.2.2.2) having the conserved amino acid residues D111, W123, D125, H126, D141 or E141, D145, K165, R194 and R199 when aligned with a pectate lyase comprising the amino acid sequence of SEQ ID NO: 2, which the variant comprises a substitution in at least one position selected from the group consisting of 5, 8, 9, 10, 19, 38, 39, 40, 41, 55, 56, 59, 61, 64, 66, 71, 72, 82, 83, 90, 100, 102, 109, 112, 114, 117, 129, 133, 136, 137, 139, 142, 144, 160, 163, 164, 166, 167, 168, 169, 171, 173, 179, 189, 192, 197, 198, 200, 203, 207, 214, 220, 222, 224, 230, 232, 236, 237, 238, 244, 246, 261, 262, 264, 265, 266, 269, 278, 282, 283, 284, 285, 288, 289 and 297, wherein the variant has pectate lyase activity. 
     
     
       2. The variant of  claim 1 , comprising a substitution at position 207. 
     
     
       3. The variant of  claim 1 , comprising a substitution at position 230. 
     
     
       4. The variant of  claim 1 , comprising a substitution at position 237. 
     
     
       5. The variant of  claim 1 , comprising a substitution at position 238. 
     
     
       6. The variant of  claim 1 , comprising a substitution at position 244. 
     
     
       7. The variant of  claim 1 , comprising a substitution at position 246. 
     
     
       8. The variant of  claim 1 , comprising a substitution at position 262. 
     
     
       9. The variant of  claim 1 , comprising a substitution at position 283. 
     
     
       10. The variant of  claim 1 , comprising a substitution at position 285. 
     
     
       11. The variant of  claim 1  comprising at least one substituted amino acid residue selected from the group consisting of A41P, T55P, V71N, S72I,T, L82I, K83N,H, W90H, L100N, I102F, G114N, L129F, L133N, D136A,P,S,T,V, F144V, V160F, G163L,H,I, M167F,I,S, L168N, M169I, E189H,N, N192Y, S197N, F198V, F200N,Y, G203V,A, N207S, S220,V, M222N,Y, N230E, L232N, A236V, K237N, D238N, Y244D, S246R,P, S261I, R262E, M265K, S261703 9P, D282H, N283P, D284P, D285G, K288P and S289P. 
     
     
       12. The variant of  claim 1 , comprising one of the following substitutions: 
       N207S; or  
       N207S+N230E; or  
       N230E.  
     
     
       13. The variant of  claim 1  comprising one of the following substitutions: 
       K237N+D238N; or  
       K237N+D238N+R262T; or  
       V244D+S246R; or  
       R262T; or  
       N283P+D285G.  
     
     
       14. A detergent composition comprising the variant of  claim 1  and a surfactant. 
     
     
       15. A method for improving the properties of cellulosic fibers, yarn, woven or non-woven fabric in which method the fibers, yarn or fabric is treated with an effective amount of the variant of  claim 1 . 
     
     
       16. The method of  claim 15 , wherein the variant is used in a scouring process step. 
     
     
       17. A method for degradation or modification of plant material in which method the plant material is treated with an effective amount of the variant of  claim 1 . 
     
     
       18. The method of  claim 17 , wherein the plant material is recycled waste paper, mechanical paper-making pulps or fibers subjected to a retting process.